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| Protocol |
DNA PREP FOR LAMBDA PHAGES |
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| AMPLIFICATION 1. Pick single plaque into 1 ml SM and soak at 370C (with shaking) for > 1 hr. 2. Plate 200-500 micoL onto single large plate; OIN 370C (use moist plates and do not invert). 3. Overlay with 12.5 ml SM plus a few drops of CHC13 and let soak >2 hrs at R.T. 4. Collect liquid which is the high titer lysate. LIOUID LYSATE 1. Innoculate 40 ml L broth + 0.2% glucose + 10 mM MgCl2 or 10 mM mgSO4 in a 250 ml erlenmeyer with: 1 ml K803 plating cells 50 ul high titer lysate (50-500 ul) for low titer lysate* A single plaque pure plug suspended in 200 ul SM. The phage and plating cells are mixed and incubated at 370C (without shaking) for 15 minutes before adding to the L broth. 2. Grow at 370C with vigorous shaking - should lyse completely in about 2-6 hours. |
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| DNA PREP 1. Add a few drops CHC13 and pellet cell debris using blue capped tubes and Sorvall bench top centrifuge or disposable 50 ml Nalgene tubes at 10 K RPM, 101, R.T. 2. Pellet phage in SW28 tubes at 23 K for >2 hrs. 3. Resuspend phage pellet - add 1.5 ml SM, break up pellet with pipet and shake gently at R.T. for about 1 hour (or overnight in refrigerator). A DNase treatment at this stage may help. 4. Set up step gradient in SW55 ultra clear tube buffer 50 mM Tris-Cl pH 8.5 2 mM MgCl2 0.1 mM EDTA 1 ml lower step = 5 M = CsCl/ml buffer " ml upper step = 3 X = CsCl/ml buffer - layer phage suspension on top - 36K 200C 601 5. Remove phage band from interface with syringe and dialyze against 0.01 M Tris pH 7.4 0.1 M NaCl 0-01 MgCl2 6. Extract: once with phenol:chloroform, once with chloroform, ethanol precipitate |
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| DNA PREP FOR LAMBDA PHAGES |
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If you want the protocol in PDF, contact Dr. Park (Your affiliation is required upon any request) |
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